Our overall objective is to elucidate the mechanism of protein biosynthesis, and, in particular, the mechanism by which initiation of translation of natural messenger RNAs occurs in eukaryotic systems. The initiation of translation of natural messenger RNAs has been extensively studied in prokaryotes. It is now known that the initiation process involves three protein factors, IF-1, IF-2, and IF-3, and the functions of these factors have been elucidated. In contrast, current evidence indicates that initiation in eukaryotes may involve as many as five or six different protein factors, and their functions are not well defined. Our first objective will be to identify and characterize the eukaryotic initiation factors from yeast and to determine their roles in the initiation of translation of synthetic and natural messengers. Yeast spheroplasts will be prepared and gently lysed in order to separate the eukaryotic protein synthesizing system from the prokaryotic system of the mitochondria. The high salt wash of the cytoplasmic ribosomes will be used as a source of the initiation factors. When the factors have been identified and partially pruified, we plan to determine how they are related functionally to initiation factors isolated from other eukaryotic and prokaryotic cells. A long-term objective is to develop a defined in vitro system for the study of eukaryotic protein biosynthesis.